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Rapid and Reliable One-Step ABO Genotyping Using Direct Real-Time Allele-Specific PCR and Melting Curve Analysis Without DNA Preparation

  • Jun-Hee Park
  • Ji-Hye Han
  • Geon ParkEmail author
Original Article
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Abstract

ABO genotyping is a molecular diagnostic technique important for transfusion and transplantation in medicine, and human identification in forensic science. Because ABO genotyping are labor intensive and time consuming, the genotyping cannot be firstly used to resolve the serological ABO discrepancy in blood bank. For rapid one-step ABO genotyping, we developed direct, real-time, allele-specific polymerase chain reaction (PCR), and melting curve analysis (DRAM assay) without DNA preparation. In DRAM assay, we used a special PCR buffer for direct PCR, a rapid RBC lysis buffer, white blood cells as template without DNA preparation, allele-specific primers for discriminating three ABO alleles (261G/del, 796C/A, and 803G/C), and melting curve analysis as a detection method. There was 100% concordance among the results of ABO genotyping by the DRAM assay, serologic typing, PCR–RFLP and PCR-direct sequencing of 96 venous blood samples. We were able to reduce the number of manual steps to three and the hands-on time to 12 min, compared to seven steps and approximately 40 min for conventional ABO genotyping using allele-specific PCR with purified DNA and agarose gel electrophoresis. We have established and validated the DRAM assay for rapid and reliable one-step ABO genotyping in a closed system. The DRAM assay with an appropriate number of allele-specific primers could help in resolving ABO discrepancies and should be valuable in clinical laboratory and blood bank.

Keywords

ABO genotyping ABO discrepancies Direct PCR Allele-specific PCR Real-time PCR Melting curve analysis 

Notes

Acknowledgments

This work was supported by a grant from the Clinical Medicine Research Institute of the Chosun University Hospital (2011).

Funding

This work was supported by a grant from the Clinical Medicine Research Institute of the University Hospital (2011).

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical Approval

All procedures performed in studies involving human blood samples were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed Consent

We obtained a waiver of informed consent from the research ethics committee on condition that we use discarded blood samples and remove all patient’s information.

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Copyright information

© Indian Society of Hematology and Blood Transfusion 2018

Authors and Affiliations

  1. 1.Department of Otolaryngology-Head and Neck SurgeryChosun University College of MedicineGwangjuSouth Korea
  2. 2.Department of Laboratory MedicineChosun University College of MedicineGwangjuSouth Korea

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